SAL (Salbutamol) ELISA Kit Catalog No: E-FS-E017 96T This manual must be read attentively and completely before using this product. If you have any problems, please contact our Technical Service Center for help. Phone: 240-252-7368(USA) Fax: 240-252-7376(USA) Email: techsupport@elabscience.com Website: Please kindly provide us the lot numbe r(on the outside of the box) of the kit for more efficient service. Copyright 2018-2019Elabscience Biotechnology Inc. All Rights Reserved
Test principle This kit uses Indirect-Competitive-ELISA as the method. It can detect SAL in samples, such as urine, tissue, feed, etc. This kit is composed of ELISA Microtiter plate, HRP conjugate, antibody, standard and other supplementary reagents. The micro-plate provided in this kit has been pre-coated with SAL. During the reaction, SAL in the samples or standard competes with SAL on the solid phase supporter for sites of SAL antibody. Then Horseradish Peroxidase (HRP) conjugate is added to each micro plate well, and TMB substrate is for color development. There is a negative correlation between the OD value of samples and the concentration of SAL. The concentration of SAL in the samples can be calculated by comparing the OD of the samples to the standard curve. Technical indicator Sensitivity: 0.1 ppb (ng/ml) Reaction mode: 25, 30 min~15 min Detection limit: Tissue/urine---0.1 ppb, Feed---1 ppb Cross-reactivity: Salbutamol---100%, Dobutamine Hydrochloride ---7.2%, Cimaterol ---0.1% Clenbuterol---3.6%, Ractopamine ---0.1%, Epinephrine ---0.1%, Isoprenaline---<1% Sample recovery rate: Urine ---90%±10%, Tissue---80%±10%, Feed---80%±15% Kits components Item ELISA Microtiter plate Standard Liquid High Concentrated Standard (100ppb) HRP Conjugate Antibody Working Solution Substrate Reagent A Substrate Reagent B Stop Solution 20 Concentrated Wash Buffer 10 Reconstitution Buffer Plate Sealer Sealed Bag Manual Specifications 96 wells 1 ml each (0ppb, 0.1ppb, 0.3ppb, 0.9ppb, 2.7ppb, 8.1ppb) 1 ml 5.5 ml 5.5 ml 40 ml 50 ml 3 pieces 1 piece 1 copy 2
Other supplies required Instruments: Microtiter plate reader, Printer, Homogenizer, Nitrogen/Water bath, Oscillators, Centrifuge, Graduated pipette, Balance (sensibility 0.01 g). High-precision transferpettor: single channel (20-200 μl, 100-1000 μl), Multichannel (300 μl). Reagents: Sodium hydroxide, Ethyl acetate, concentrated HCl, Acetonitrile, Isopropanol, N-hexane, Anhydrous sodium sulphate, Methanol. Experimental preparation Bring all reagents and samples to room temperature before use. Open the micro-plate reader in advance, preheat the instrument, and set the testing parameters. 1. Sample pretreatment Notice: Experimental apparatus should be clean, and the pipette should be disposable to avoid crosscontamination during the experiment. 2. Solution preparation Solution 1: 1 M HCl Solution Dissolve 8. of concentrated HCl to 100 ml with deionized water Solution 2: 1 M NaOH Solution Dissolve 4 g of NaOH to 100 ml with deionized water Solution 3: Reconstitution Buffer Dilute the 10 Reconstitution Buffer with deionized water. (2 Reconstitution Buffer (V): Deionized water (V)=1:9).The Reconstitution buffer can be store at 4 for a month. Solution 4: Wash Buffer Dilute 20 Concentrated Wash Buffer with deionized water. (20 Concentrated Wash Buffer (V): Deionized water (V) = 1:19) 3. Sample pretreatment procedure 3.1 Pretreatment of urine: Take 50 μl of clear urine sample to detect (the turbid urine sample should be filtered or centrifuge at 4000 r/min for 5 min to get clear urine sample). Temporarily used samples should be kept frozen to save. Note: Sample dilution factor: 1, minimum detection dose: 0.1 ppb 3.2 Pretreatment 1 of tissue: Weigh 2±0.05 g of homogeneous tissue samples, add of Reconstitution buffer, oscillate thoroughly for 2 min, centrifuge at 4000 r/min at room temperature for 10 min (if the lipid content of tissue samples is high, it s suggested to incubate at 85 with water bath for 10 min after oscillating, then centrifuge). Take 50 μl of upper liquid to analyze. Note: Sample dilution factor: 4, minimum detection dose: 0.4 ppb 3
3.3 Pretreatment 2 of tissue: (1) Weigh 2±0.05g of homogeneous tissue samples,, add 1 ml of 1 Reconstitution solution, oscillate thoroughly, add 4 ml of acetonitrile, oscillate thoroughly, add 1 ml of isopropanol, oscillate thoroughly for 5 min, centrifuge at 4000 r/min at room temperature for 10 min. (2) Take 3 ml of upper liquid, add 50 μl of 1 M NaOH, add 7 ml of Ethyl acetate, oscillate thoroughly for 5 min, centrifuge at 4000 r/min at room temperature for 10 min. Take all upper liquid to blowdry at 56 with nitrogen or water bath. (3) Dissolve the dried residual with 1 ml of Reconstitution buffer, add 1 ml of n-hexane, mix 30 s; centrifuge at 4000 r/min at 15 for 5 min. (4) Take 50 μl of lower liquid to analyze. Note: Sample dilution factor: 1, minimum detection dose: 0.1 ppb 3.4 Pretreatment of Feed: (1) Weigh 1±0.05 g of homogeneous Feed sample, add 10 ml of Methanol and 5g of Anhydrous sodium sulphate, oscillate for 2 min, Centrifuge at 4000 r/min at room temperature for 10 min. (2) Take 1 ml of upper liquid after centrifugation, blow-dry at 56 with nitrogen or water bath, dissolve the dried residual with 1 ml of Reconstitution buffer, add 1 ml of n-hexane, mix 30 sec; Centrifuge at 4000 r/min at room temperature for 5 min. (3) Take 50 μl of lower liquid to analyze. Note: Sample dilution factor: 10, minimum detection dose: 1 ppb Assay procedure Centrifuge the sample again after thawing before the assay. Bring all reagents to room temperature before use. All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. 1. Number: number the sample and standard in order (multiple well), and keep a record of standard wells and sample wells. 2. Add sample: add 50 μl of Standard or Sample per well, then add 50 μl of HRP Conjugate to each well, then add 50 μl of antibody working solution, cover the plate with sealer we provided, oscillate for 5 sec gently to mix thoroughly, incubate for 30 min at 25. 3. Wash: uncover the sealer carefully, remove the liquid in each well. Immediately add 300 μl of wash buffer to each well and wash. Repeat wash procedure for 5 times, 30 sec intervals/time. Invert the plate and pat it against thick clean absorbent paper (If bubbles exist in the wells, clean tips can be used to prick them). 4. Color Development: add 50 μl of substrate solution A to each well, and then add 50 μl of substrate solution B. Gently oscillate for 5 sec to mix thoroughly. Incubate shading light for 15 min at 25. 5. Stop reaction: add 50 μl of stop solution to each well, oscillate gently to mix thoroughly. 6. OD Measurement: determine the optical density (OD value) of each well at 450 nm with a microplate reader (the 450/630 nm double wavelength is recommended). This step should be finished in 10 min after stop reaction. 4
Result analysis 1. Absorbance (%)=A/A 0 100% A: Average absorbance of standard or sample A 0: Average absorbance of 0 ppb Standard 2. Drawing and calculation of standard curve Create a standard curve by plotting the absorbance percentage of each standard on the y-axis against the log concentration on the x-axis to draw a semi-logarithmic plot. Add average absorbance value to standard curve to get corresponding concentration. If samples have been diluted, the concentration calculated from the standard curve must be multiplied by the dilution factor. For this kit, it is more convenient to use professional analysis software for accurate and fast analysis on a large number of samples. Notes 1. Overall OD value will be lower when reagents is not brought to room temperature before use or room temperature is below 25. 2. During the washing procedure, if the wells turn dry, it will lead to bad linear standard curve and poor repeatability, move on to the next step immediately after wash. 3. Mix thoroughly and wash the plate completely. The consistency of wash procedure can strongly affect the reproducibility of this ELISA kit. 4. ELISA Microtiter plate should be covered by plate sealer. Avoid the reagents to strong light. 5. Do not use expired kit and reagents of different batches. 6. TMB should be abandoned if it turns color. When OD value of standard(concentration: 0)<0.5 unit(a 450nm<0.5), it indicates reagent is deteriorated. 7. Stop solution is caustic, avoid contact with skin and eyes. Storage and valid period Storage: Store at 2-8. Avoid freeze / thaw cycles. Valid Period: 1 year, expiration date is on the packing box. Copyright 2018-2019Elabscience Biotechnology Inc. All Rights Reserved 5